Cancer is a major disease burden and one hope of changing this is to develop new treatments through a[unreadable] better understanding of the disease. Recent discoveries concerning the activities of short RNAs in mammals[unreadable] may provide both new insights and new treatments of cancer. A common goal of this Program is to[unreadable] investigate the roles of short RNAs, such as microRNAs, in regulation of genes in normal and cancer cells.[unreadable] There is strong and rapidly growing evidence suggesting that changes in miRNA regulation are related to[unreadable] malignant transformation and in fact could be a critical event in oncogenic transformation. The function of[unreadable] the mir-17-92-1 cluster which is frequently overexpressed/amplified in a subset of human cancers will be[unreadable] investigated by creation of specific mutations of these microRNAs in the context of mouse models of cancer.[unreadable] Changes in microRNA populations in normal cells and tumor cells of the same developmental state will be[unreadable] analyzed using both bead-array technology as well as new cloning technology. Vectors with regulated[unreadable] expression of a short hairpin RNA which generates a specific siRNA for silencing a gene will be developed[unreadable] for transgenic analysis of pathways. Additionally, methods will be tested for screening of small libraries of[unreadable] shRNA-lentiviral vectors to identify genes which, when silenced, either inhibit or stimulate tumor[unreadable] development. Furthermore, libraries of retro viral vectors expressing shRNAs will be used in screens to[unreadable] identify (a) genes that modulate the proliferation and/or survival of pRB-deficient cells , (b) genes that[unreadable] modulate the rate of development of a K-ras-driven lung cancer model, and (c) genes important for the[unreadable] differentiation of ES cells. The potential role of short RNAs in transcriptional silencing will be investigated in[unreadable] embryonic stem cells. These processes could be important for epigenetic silencing and genomic stability of[unreadable] cancer cells. ES cells will also be studied for the role of miRNAs in development and proliferation. Changes[unreadable] in the spectrum of microRNAs and siRNAs during T-cell development will be characterized using a cloning[unreadable] technology which requires small amounts of RNAi. Activation of the Arf promoter is an early signal in[unreadable] oncogenic transformation. This promoter is silenced under normal conditions by the E2F3B protein, linking[unreadable] the p19Arf-mdm2-p53 pathway to the p16INK4a-cycD/cdk4-pRB-EdF pathway. The role of E2F3B complexes[unreadable] and other E2F factors in regulation of the Arf promoter will be studied.